PROJECT SUMMARY Metastatic melanoma ranks among the most lethal cancers, and while recent clinical advances have led to curative treatments in some patients, there remains a dire need for improved therapeutic strategies with better survival outcomes. A detailed molecular understanding of melanomagenesis is paramount to the development of such strategies. Over the last three decades genetically engineered mouse models (GEMMs) have become an invaluable research tool to characterize gene functions in disease, to recapitulate the genetics and etiology of human malignancies, and to preclinically evaluate novel cancer therapies. Indeed, GEMMs have demonstrated the causal involvement of genetic abnormalities found in melanoma patients, for instance activating mutations in BRAF or genomic loss of PTEN. Despite the wealth of information that can be gained from mouse models, they have certain disadvantages that often limit their utility for cancer research. Chief among these disadvantages are the lengthy and inefficient process of generating new mouse strains and the time-consuming breeding of compound mutant experimental mice. We have generated an embryonic stem cell-GEMM (ESC-GEMM) platform that will significantly enhance the rate and throughput of in vivo melanoma research. This approach relies on newly derived ESCs that harbor several alleles for i) the initiation of melanoma, ii) the activation of Tet-inducible expression cassettes, and iii) the efficient delivery of such expression cassettes via recombination-mediated cassette exchange (RMCE). Importantly, when combined with novel genetic tools such as inducible shRNAs or CRISPR/Cas9, these ESC lines will provide a versatile resource to interrogate fundamental unanswered questions in melanoma biology. This proposal is focused on the in vivo validation of this ESC-GEMM resource. First, we will determine if mice generated with the ESC- GEMM approach develop melanoma driven by oncogenic Braf and loss of Pten with kinetics similar to mice that were generated by conventional breeding. Second, we will perform a proof-of-principle experiment to demonstrate the compatibility of the ESC-GEMM platform with various methods of abolishing gene expression. Specifically, we will compare how potently depletion of Pten by traditional conditional knockout, shRNA, or CRISPR/Cas9 cooperates with BrafV600E to drive melanoma formation. The analyses outlined in this proposal will validate the functionality and utility of our ESC-GEMM platform, which we will use extensively in the future to study drivers of melanomagenesis. Importantly, once validated, we will make this resource freely available to the scientific community to stimulate research into the causes and vulnerabilities of melanoma.